Regulation of GPCR Recycling at the Plasma Membrane

质膜上 GPCR 回收的调控

• 批准号: 7456554
• 负责人: Guillermo Ariel Yudowski
• 金额: $ 8.76万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7456554
• 关键词: ADRB2 gene; Adrenergic Receptor; Agonist; Amino Acid Sequence; Appendix; Area; Award; Behavior; Cell Surface Receptors; Cell membrane; Cell model; Cell surface; Cells; Characteristics; Complex; Docking; Endocytosis; Event; Family; Frequencies; G-Protein-Coupled Receptors; Image; Institution; Laboratories; Lateral; Life; Ligands; Measurement; Mediating; Membrane Proteins; Molecular; Nature; Neuraxis; Neurons; Numbers; Opioid Receptor; Pathologic Processes; Pathway interactions; Pharmaceutical Preparations; Physiological; Positioning Attribute; Process; Proteins; Receptor Activation; Receptor Signaling; Recycling; Regulation; Relative (related person); Research; Signal Transduction; Sorting - Cell Movement; Specificity; Stimulus; Tail; Techniques; Testing; Therapeutic; Thinking; Total Internal Reflection Fluorescent; Universities; Vesicle; Work; beta-2 Adrenergic Receptors; drug of abuse; genetic regulatory protein; insight; mu opioid receptors; novel strategies; receptor; receptor recycling; response

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCR) are integral plasma membrane proteins that transduce external signals into the cell. GPCRs mediate many important signaling functions in the central nervous system (CMS) and mediate the effects of many therapeutic and abused drugs. A basic mechanism by which these receptors are regulated is by ligand-induced endocytosis. This process reduces the number of activated receptors at the cell surface therefore regulating receptor signaling. While endocytosis is strongly regulated by physiological ligands and drugs, it is generally believed that recycling is a nonregulated process. New evidence from our laboratory and others are challenging this default hypothesis of GPCR recycling. We have developed a technique to investigate single insertion events at the cell surface by combining live total reflection microscopy (TIRF) with a pH sensitive extracelullar tag fused to the extracelullar terminus of GPCRs. Our preliminary results indicate that receptor recycling is a tightly regulated process involved in the homeostatic response to extracelullar stimuli. Furthermore, regulation of recycling is dependent on receptor activation, which ultimately decreases the frequency of recycling events reducing receptor number at the cell surface. I propose to extend and further develop this novel approach to examine: a) The characteristics of the vesicles mediating receptor recycling, b) Identify the machinery involved in the fusion process, and its possible regulation by receptor activity, c) Investigate if the regulation of receptor recycling is a general process for GPCRs, d) Define and investigate the functionality of newly inserted receptors and the possible existence of a molecular complex associated to reinserted receptors. This work will be perfomed using an experimentally advantageous heterologous cell model, and the results will be later examined in hippocamapal neurons. By investigating the regulation of receptor insertion to the cell surface, our work has the potential to provide fundamental new insight to key cellular events likely to be relevant to a wide variety of physiological and pathological processes. Obtaining the K99/ROO award will be an extraordinary opportunity to transition from my postdoctoral studies to an independent position in a university/research institution.

描述（由申请人提供）：G蛋白偶联受体（GPCR）是将外部信号转导至细胞内的完整质膜蛋白。 GPCR 介导中枢神经系统 (CMS) 中许多重要的信号传导功能，并介导许多治疗药物和滥用药物的作用。调节这些受体的基本机制是配体诱导的内吞作用。这个过程减少了细胞表面激活受体的数量，从而调节受体信号传导。虽然内吞作用受到生理配体和药物的强烈调节，但通常认为回收是一个不受调节的过程。来自我们实验室和其他实验室的新证据正在挑战 GPCR 回收的这一默认假设。我们开发了一种技术，通过将实时全反射显微镜 (TIRF) 与融合到 GPCR 细胞外末端的 pH 敏感细胞外标签相结合，研究细胞表面的单次插入事件。我们的初步结果表明，受体再循环是一个严格调控的过程，涉及对细胞外刺激的稳态反应。此外，再循环的调节取决于受体激活，这最终降低了再循环事件的频率，从而减少了细胞表面的受体数量。我建议扩展和进一步开发这种新方法来检查：a）介导受体再循环的囊泡的特征，b）识别融合过程中涉及的机制，及其可能受受体活性的调节，c）研究受体再循环的调节是否是GPCR的一般过程，d）定义和研究新插入受体的功能以及与重新插入相关的分子复合物的可能存在 受体。这项工作将使用实验上有利的异源细胞模型进行，随后将在海马神经元中检查结果。通过研究受体插入细胞表面的调节，我们的工作有可能为可能与多种生理和病理过程相关的关键细胞事件提供基本的新见解。获得 K99/ROO 奖将是我从博士后研究过渡到大学/研究机构独立职位的绝佳机会。

项目成果

Imaging of kiss-and-run exocytosis of surface receptors in neuronal cultures.

• DOI: 10.3389/fncel.2014.00363
• 发表时间: 2014
• 期刊: Frontiers in cellular neuroscience
• 影响因子: 5.3
• 作者: Roman-Vendrell C;Chevalier M;Acevedo-Canabal AM;Delgado-Peraza F;Flores-Otero J;Yudowski GA
• 通讯作者: Yudowski GA

Acute inactivation of PSD-95 destabilizes AMPA receptors at hippocampal synapses.

PSD-95 的急性失活会破坏海马突触 AMPA 受体的稳定性。

• DOI: 10.1371/journal.pone.0053965
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Yudowski,GuillermoA;Olsen,Olav;Adesnik,Hillel;Marek,KurtW;Bredt,DavidS
• 通讯作者: Bredt,DavidS