Spatiotemporal Regulation of GPCR Signaling by Different Beta-Arrestin Conformations

不同 Beta-arrestin 构象对 GPCR 信号传导的时空调节

• 批准号: 10682474
• 负责人: Alex Rojas Bie Thomsen
• 金额: $ 40.24万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10682474
• 关键词: Agonist; Arrestins; Binding; Binding Sites; Biochemical; Biological; C-terminal; Cell surface; Cells; Complex; Coupling; Cyclic AMP; Data; Drug Prescriptions; Drug Targeting; Endocytosis; Endosomes; Future; G Protein-Coupled Receptor Signaling; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GTP-Binding Protein Regulators; GTP-Binding Proteins; Heterotrimeric GTP-Binding Proteins; Hydrophobicity; Knowledge acquisition; Location; Mediating; Membrane; Molecular; Molecular Conformation; Phosphorylation; Phosphorylation Site; Physiological; Physiological Processes; Property; Proteomics; Regulation; Research; Signal Transduction; Signaling Protein; Tail; Therapeutic; Time; Vision; beta-arrestin; desensitization; design; driving force; drug development; innovation; phosphoric diester hydrolase; programs; receptor; receptor internalization; recruit; response; spatiotemporal

G protein-coupled receptors (GPCRs) at the cell surface regulate most physiological processes and are important drug targets with ~34% of all prescribed drugs targeting them. Classically, upon agonist stimulation, GPCRs activate heterotrimeric G proteins, causing downstream signaling throughout the cell. In order to terminate G protein signaling, cells have devised a specialized desensitization mechanism that includes receptor phosphorylation by GPCR kinases and subsequent recruitment of β-arrestins (βarrs) to the phosphorylated receptors. The GPCR–βarrs interaction both blocks the G protein-binding site at the receptor core and promotes receptor endocytosis. Recently, however, we discovered that some GPCRs interact with βarrs exclusively through their phosphorylated C-terminal tails. Since βarrs do not block the G protein-binding site in this `tail' conformation, the receptor can associate with βarrs and G proteins simultaneously to form GPCR–G protein– βarr `megaplexes.' The assembly of these megaplexes allows the receptor to continue to stimulate G protein signaling while being internalized into endosomes by βarrs. Thus, the existence of the core and tail GPCR–βarr complex conformations suggests that βarrs act as spatiotemporal master regulators of G protein signaling: When bound to the receptor core, βarrs regulate the duration of G protein signaling whereas βarrs control the cellular location from where G proteins are activated from when associated with the receptor C-terminal tail. As the underlying properties that promote these two complex conformations remain elusive, my research objectives over the next 5 years involve determining these molecular driving forces on a general scale. Our preliminary data suggest that phosphorylation site clusters located within the receptor C-terminal tail are required for the association with βarrs in the tail conformation. Therefore, we plan to establish whether the presence of these phosphorylation site clusters correlates with the capacity of GPCRs to engage in mechanisms that lead to sustained endosomal G protein signaling. In regards to the GPCR–βarr core conformation, the fingerloop domain (FLD) of βarrs inserts itself into the transmembrane core of most GPCRs via its hydrophobic tip and receptor- specific residues. To characterize this interaction on a general scale, we will examine whether these receptor- specific βarr-FLD residues correlate with G protein subtype coupling of different GPCRs. Finally, βarrs modulate the activity of phosphodiesterases (PDEs), which terminate Gs-cAMP signaling. However, our preliminary data raise the possibility that this modulation occurs specifically by βarrs in the core conformation. Therefore, we will apply a combination of cell biological, biochemical, and proteomics approaches to examine whether modulation of PDEs and other desensitization mechanism is mediated specifically by distinct βarr conformations. My vision with this research program is to elucidate how GPCR signaling is regulated spatiotemporally by βarrs, which may lead to differentiated physiological responses. The knowledge acquired here will be used to design new and innovative therapeutics that specifically target GPCRs in time and space.

细胞表面的G蛋白偶联受体（GPCR）调节大多数生理过程， 重要的药物靶点，约34%的处方药以它们为靶点。传统上，在激动剂刺激下， GPCR激活异源三聚体G蛋白，引起整个细胞的下游信号传导。为了 终止G蛋白信号，细胞已经设计了一种专门的脱敏机制，包括受体 通过GPCR激酶的磷酸化和随后的β-抑制蛋白（βarrs）向磷酸化的 受体。GPCR-βarrs相互作用既阻断了受体核心的G蛋白结合位点，又促进了GPCR-β arrs相互作用。 受体内吞作用然而，最近我们发现一些GPCR仅与βarrs相互作用， 通过它们磷酸化的C-末端尾部。由于βarrs不阻断G蛋白结合位点在这条“尾巴”上， 构象，受体可同时与βarrs和G蛋白结合形成GPCR-G蛋白。 β-葡糖苷酶。这些megaplexes的组装允许受体继续刺激G蛋白 信号传导，同时被βarrs内化到内体中。因此，核心和尾部GPCR-β的存在 复杂的构象表明，βarrs作为G蛋白信号传导的时空主调节器： 结合到受体核心，βarrs调节G蛋白信号传导的持续时间，而βarrs控制细胞内的 当与受体C-末端尾结合时，G蛋白被激活的位置。为 促进这两种复杂构象的基本性质仍然难以捉摸，我的研究目标是 未来5年的研究涉及在一般范围内确定这些分子驱动力。我们的初步数据 这表明位于受体C-末端尾内的磷酸化位点簇是蛋白质合成所必需的。 在尾部构象中与βarrs结合。因此，我们计划确定是否存在这些 磷酸化位点簇与GPCR参与机制的能力相关， 持续的内体G蛋白信号传导。关于GPCR-β-D核心构象，指环结构域 (FLD)的βarrs通过其疏水末端和受体插入大多数GPCR的跨膜核心， 具体的残留物。为了在一般尺度上描述这种相互作用，我们将研究这些受体- 特异性βarr-FLD残基与不同GPCR的G蛋白亚型偶联相关。最后，βarrs调节 终止Gs-cAMP信号传导磷酸二酯酶（PDE）的活性。然而，我们的初步数据显示， 提出了这种调节通过核心构象中的βarrs特异性发生的可能性。所以我们会 应用细胞生物学，生物化学和蛋白质组学方法的组合来检查是否调节 PDEs和其他脱敏机制是由不同的β-环糊精构象特异性介导的。我的视力 本研究旨在阐明βarrs如何在时空上调节GPCR信号， 可能导致不同的生理反应。在这里获得的知识将用于设计新的和 在时间和空间上特异性靶向GPCR的创新疗法。

项目成果

β-arrestin-dependent and -independent endosomal G protein activation by the vasopressin type 2 receptor.

β-arrestin 依赖和独立的内体 G 蛋白由加压素 2 型受体激活。

• DOI: 10.1101/2023.04.01.535208
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Daly,Carole;Guseinov,AkimAbdul;Hahn,Hyunggu;Wright,Adam;Tikhonova,IrinaG;Thomsen,AlexRojasBie;Plouffe,Bianca
• 通讯作者: Plouffe,Bianca